Hermann M. Wolf

Prevention of Necrotizing Enterocolitis in Low-Birth-Weight Infants by IgA–IgG Feeding

In a randomized clinical trial, we evaluated the efficacy of an oral immunoglobulin preparation (73 percent IgA and 26 percent IgG) in reducing the incidence of necrotizing enterocolitis in infants of low birth weight for whom breast milk from their mothers was not available. A total of 434 infants weighing between 800 and 2000 g were eligible for entry in the study. Of these, 255 were withdrawn - 234 during the first week of the study because breast milk from their mothers became available (123 in the treatment group and 111 in the control group), and 21 because of violations of protocol or because breast milk became available after the first week. The duration of follow-up was 28 days. Among the infants for whom breast milk did not become available during the study, there were no cases of necrotizing enterocolitis among the 88 receiving oral IgA-IgG, as compared with six cases among the 91 control infants (P = 0.0143). Of the infants withdrawn from the study, two assigned to the control group had necrotizing enterocolitis. We conclude that the oral administration of IgA-IgG may prevent the development of necrotizing enterocolitis in low-birth-weight infants.

Results of a prospective controlled two-dose crossover study with intravenous immunoglobulin and comparison (retrospective) with plasma treatment☆

Results of a two-dose (150 vs 500 mg/kg/month) crossover study with intravenous immunoglobulin (Endobulin, Immuno) (IVIG) carried out on 12 children with primary immunodeficiency syndromes over a period of 2 years are reported. Eight children had received human plasma (20 mg/kg/month) during the 2 years prior to the IVIG study. As these children had been thoroughly monitored during plasma treatment, a retrospective analysis of these data allowed for comparison with IVIG treatment. Children on low-dose IVIG therapy had significantly (P less than 0.01) fewer days with clinical illness, e.g., sinusitis, pneumonia, diarrhea, and arthritis, than did those receiving plasma treatment. High-dose IVIG therapy led to further significant clinical improvement. Lung function tests (MEF25) improved significantly as well. The difference between high- and low-dose therapy with respect to the improvement in clinical symptoms (e.g., cold, fever, otitis) was more pronounced in children with severe clinical symptoms at the initiation of the study. Children with fewer symptoms did comparably well on high- and low-dose treatment, except for those with acute febrile illness, which was less frequent in children on high-dose IVIG. Regular monitoring of liver enzymes in the group of patients on IVIG therapy gave no indication of the transmission of viral hepatitis in the course of the 2-year IVIG treatment.

Reassessment of autoreactivity of the broadly neutralizing HIV antibodies 4E10 and 2F5 and retrospective analysis of clinical safety data

Background:The broadly neutralizing recombinant human HIV-1 antibodies 4E10, 2F5 and Igh1b12 are reported to have autoreactive potential, which is significant for HIV-1 vaccine development and passive immunotherapy using these antibodies. Objective:To investigate the clinical relevance of these findings in subjects receiving passive immunotherapy with these antibodies. Methods:Four types of investigations were performed: (1) Investigation of clotting parameters in an ongoing clinical study with 4E10, 2F5 and 2G12. (2) Mixing experiments of pooled plasma with the same antibodies. (3) Retrospective analysis of serum from patients who received passive immunotherapy with 4E10, 2F5 and 2G12 either alone or in combination. (4) Assessment of clinical safety data obtained after 418 infusions with these antibodies. Results:Standard clinical assays confirmed that 4E10 showed low-level cross-reactivity with cardiolipin, while previously reported cardiolipin cross-reactivity for 2F5 could not be confirmed. High serum titers of 4E10 induced mild prolongation of the activated partial thromboplastin time, which resolved with the wash out of 4E10. Neither 2F5 nor 2G12 affected coagulation. Repeated high-dose infusions of the monoclonal antibody combination were well tolerated with no incidence for thrombotic complications after 418 infusions in 39 subjects. Conclusions:Monoclonal antibody 4E10 but not 2F5 or 2G12 showed autoreactive binding specificities. Infusion of 4E10 resulted in transient low anticardiolipin titers. Although an increased thromboembolic risk cannot definitely be excluded, this risk appears to be low and likely depend on underlying disorders.

Regulation of TCR-mediated T cell activation by TNF-RII

In the present study, we investigated the role of tumor necrosis factor receptor II (TNF‐RII) in human T cell activation induced via the T cell receptor (TCR) in an antigen‐presenting cell‐independent system. Our results confirm that interaction of TNF‐α with TNF‐RII but not TNF‐RI is directly costimulatory to TCR‐mediated T cell activation, thereby augmenting T cell proliferation, expression of T cell activation markers (CD25, human leukocyte antigen‐DR, TNF‐RII), and secretion of cytokines such as interferon‐γ and TNF‐α. In contrast to the well‐defined costimulatory molecule CD28, costimulation via TNF‐RII showed significant differences in kinetics, requirement for cross‐linking, redundancy of intracellular signaling pathways involved, and the capacity to induce interleukin (IL)‐2, IL‐10, and IL‐13 secretion. In addition, cross‐linking TNF‐RII had the capacity to down‐regulate TCR/CD28‐induced Ca++ mobilization, IL‐2 mRNA expression, and IL‐2 and IL‐10 secretion. Taken together, our findings demonstrate that TNF‐RII plays a unique role among the T cell costimulatory molecules, as TNF‐RII ligation can have positive and negative effects on TCR‐dependent signaling. TNF‐RII cross‐linking has an inhibitory effect on early TCR signaling events proximal to induction of Ca++ flux, which ultimately leads to modulation of the T cell cytokine pattern expressed.

Long‐term decrease of CD4+CD45RA+ T cells and impaired primary immune response after post‐traumatic splenectomy

Congenital or acquired absence of the spleen and functional hyposplenism are associated with abnormalities of host defence such as an increased susceptibility to infection with encapsulated bacteria. The effects of the lack of the spleen on cell‐mediated immunity are largely unknown. In the present study we have investigated peripheral blood lymphocyte subpopulations in healthy adults who had undergone splenectomy because of severe abdominal trauma > 4 years before the study. The results show a significant reduction in the percentage of CD4+ T cells due to a selective and long‐term decrease in the percentage of CD4+CD45RA+ lymphocytes, the CD4+ T‐cell subset mainly involved in primary immune responses to newly encountered antigens. Levels of the reciprocal CD45RO+CD4+ T‐cell subset were comparable between splenectomized and control individuals, as were lymphoproliferative responses and IFN‐gamma production to recall antigens. Decreased levels of CD4+CD45RA+ cells were accompanied by an impairment in primary immune responsiveness, as assessed by investigating T‐cell proliferation to stimulation with keyhole limpet haemocyanin and by measuring antibody responses following primary immunization with a clinically relevant T‐dependent antigen, hepatitis A vaccine, in vivo. These findings suggest a possible role of the spleen in the generation, maintenance and/or differentiation of naive, unprimed T cells or their precursors, which might have a possible functional relevance for primary immune responses following splenectomy.

Inhibition of Receptor-Dependent and Receptor-Independent Generation of the Respiratory Burst in Human Neutrophils and Monocytes by Human Serum IgA

ABSTRACT: An important feature of the role of IgA in protection against infection and disease at the level of the mucosal surfaces might be the elimination of pathogens without induction of a strong inflammatory reaction. In the present study we addressed the question whether IgA has a regulatory effect on the generation of reactive oxygen intermediates in human neutrophils and monocytes (i.e. the respiratory burst). Cells were stimulated with heat-inactivated Haemophilus influenzae type b or phorbol myristate acetate, stimuli known to use different recognition structures or signal transduction pathways. Concentrations of IgA as low as 10 mg/L significantly inhibited the receptor-dependent Haemophilus influenzae-induced respiratory burst in granulocytes, as assessed by measuring luminol-enhanced chemiluminescence. Furthermore, IgA had a dose-dependent inhibitory effect on the receptor-independent induction of the respiratory burst, as examined by flow cytometry in monocytes and granulocytes activated with phorbol myristate acetate. Our results therefore indicate that inhibition of receptor-ligand interaction is not a sufficient explanation for the IgA-mediated modulation of the respiratory burst in human phagocytic cells. In addition, IgA might directly regulate the activation of the respiratory burst at the level or downstream of protein kinase C activation. By modulating the release of mediators of inflammation such as reactive oxygen intermediates, the inflammatory response could be down-regulated at the level of the mucosal surfaces, thereby preventing the development of sequelae of an exaggerated inflammatory response potentially leading to local or systemic pathology.

Double mutant and formaldehyde inactivated TSST-1 as vaccine candidates for TSST-1-induced toxic shock syndrome

Up to now there is no treatment for staphylococcal toxic shock syndrome, a disease mainly induced by toxic shock syndrome toxin-1(TSST-1). There is great demand in finding means to control the disease, one of them is the development of an effective and safe vaccine against TSST-1. In this study we constructed a series of vaccine candidates and investigated their biological activity, toxicity, and potential to invoke an immune response. TSST-1 was isolated from Stahylococcus aureus supernatants and recombinantly expressed as a N-terminal 6x histidine-tagged protein in Escherichia coli. In order to obtain molecules with minimal toxicity we constructed single mutants (G31R and H135A) and one double mutant (G31R/H135A) with both residues exchanged. We also detoxified native TSST-1 isolated from S. aureus, and recombinantly expressed TSST-1 by treatment with formaldehyde. Functional activity of native and recombinant TSST-1 and grade of inocuity of mutants and toxoids was determined by investigating mitogenity, T-cell activation, and cytokine release upon stimulation of human mononuclear cells with the vaccine candidates. All substances were tested in a rabbit immunization study. After primary immunization and three additional boosts all vaccinated animals developed antibody titers against TSST-1 and were protected against challenge with a lethal doses of superantigen potentiated with lipopolysaccharide.

The anti‐inflammatory effect of an oral immunoglobulin (IgA‐IgG) preparation and its possible relevance for the prevention of necrotizing enterocolitis

An exaggerated release of inflammatory mediators has been implicated in the pathogenesis of necrotizing enterocolitis (NEC). Oral administration of a human immunoglobulin preparation (serum IgA‐IgG) has been demonstrated to be an effective prophylaxis for NEC. The aim of the present study was to examine the regulatory effect of a human IgA‐IgG preparation on the release of inflammatory cytokines in human monocytes. Our results indicate that the immunoglobulin preparation inhibits TNF‐alpha and IL‐6 release in monocytes following stimulation with heat‐inactivated Hib in a dose‐dependent manner. This might have a biological relevance in infants receiving oral immunoglobulin prophylaxis for NEC, since modulation of the release of inflammatory mediators at the level of the gastrointestinal mucosa could interfere with the development of noxious sequelae of acute and/or chronic inflammation initiated by microbial pathogens or their toxins that finally lead to the pathologic changes associated with NEC.

Effect of Polymeric IgG on Human Monocyte Functions

Fc and iC3b receptors are involved in various biological functions of phagocytic cells, such as immune adherence and phagocytosis of opsonized particles, degranulation and superoxide generation. In the present study we examined the expression of specific receptors for the Fc portion of IgG (FcR) and for iC3b (CR3), a cleavage product of the third complement component, on human monocytes following in vitro treatment with polymeric and monomeric IgG. Interaction of polymeric IgG (fluid phase) with the monocyte membrane led to a concomitant modulation of both Fc and iC3b receptors. Monomeric IgG, however, down modulated Fc receptor expression only if surface bound. Under these conditions, no concomitant modulation of the iC3b receptor could be observed. The down modulation of Fc and iC3b receptors induced by fluid-phase IgG polymers was also accompanied by a decrease in monocyte functions as expressed by reduced Fc receptor-mediated phagocytosis, decreased release of oxygen metabolites following stimulation by aggregated IgG and opsonized zymosan, as well as in impaired killing of bacteria. These data suggest that a down modulation of Fc and iC3b receptors might have important implications for host defense mechanisms, since interaction with these receptors is required for the proper elimination of many pathogens.

Mutation in a winged-helix DNA-binding motif causes atypical bare lymphocyte syndrome

Bare lymphocyte syndrome (BLS) is an autosomal recessive severe-combined immunodeficiency that can result from mutations in four different transcription factors that regulate the expression of major histocompatibility complex (MHC) class II genes. We have identified here the defective gene that is responsible for the phenotype of the putative fifth BLS complementation group. The mutation was found in the regulatory factor that binds X-box 5 (RFX5) and was mapped to one of the arginines in a DNA-binding surface of this protein. Its wild-type counterpart restored binding of the RFX complex to DNA, transcription of all MHC class II genes and the appearance of these determinants on the surface of BLS cells.